The research described in this proposal is directed towards understanding the mechanism of asparagine- linked glycosylation of proteins in the endoplasmic reticulum (ER) by the oligosaccharyltransferase (OST). Particular emphasis will be placed upon (i) the generation and analysis of HEK293 derived cell lines that do not express specific subunits of the STT3A or STT3B complexes, (ii) the analysis of the role of the STT3A complex in cotranslational glycosylation of cysteine-rich glycoproteins, (iii) elucidation of the molecular mechanism responsible for localization of the STT3A complex adjacent to the protein translocation channel and (iv) analysis of the mechanism of recognition of nonglycosylated acceptor sites in proteins by the STT3B complex. Current evidence indicates that the STT3A isoform of the OST is primarily responsible for cotranslational N-glycosylation of nascent polypeptides as they pass through the protein translocation channel. The STT3B isoform can mediate posttranslocational glycosylation of acceptor sites that have been skipped by the STT3A complex prior to folding of the nascent glycoprotein. The CRISPR-Cas9 gene- editing system will be used to generate HEK293 derived cell lines that lack STT3A, STT3B or accessory subunits of the STT3A or STT3B complexes. Mass spectrometry will be used to catalogue the HEK293 glycoproteome and to identify glycosylation acceptor sites that are hypoglycosylated when cells lack a functional STT3A or STT3B complex. Glycopeptide sequence analysis should identify motifs in glycosylation sites that promote skipping by the STT3A complex, and indicate what fraction of the total acceptor sites are modified by each OST isoform. Cysteine rich glycoproteins like prosaposin and progranulin are hypoglycosylated when the STT3A complex is depleted by siRNA treatment. Bioinformatics analysis will be used to identify cysteine-rich glycoproteins in model organisms and to determine the spacing between glycosylation acceptor sites and cysteine residues. In vivo pulse labeling in wild type and mutant cells will be used to determine whether the STT3A complex is responsible for glycosylation of proteins that are comprised of multiple cysteine-rich repeat domains. Large complexes consisting of the STT3A complex, the protein translocation channel (Sec61 complex) and the translocon- associated protein (TRAP) complex have been detected by native gel electrophoresis. The role of the TRAP complex and role of STT3A-specific accessory subunits in the assembly and stability of these large complexes will be explored to determine how the STT3A complex is positioned adjacent to the protein translocation channel. Co-immunoprecipitation experiments will be performed to determine whether binding of hypoglycosylated proteins to the STT3B isoform of the OST is mediated by the MagT1 or TUSC3 oxidoreductases. Together, this research will help reveal how cooperation between the STT3A and STT3B complexes enhances the glycosylation efficiency of proteins in human cells.